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Keywords:

  • Lung transplant;
  • vWF;
  • xenograft

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Porcine von Willebrand factor (vWF) activates human and primate platelets. Having determined the importance of pulmonary intravascular macrophages (PIMs) in pulmonary xenotransplantation, we evaluated whether, in the absence of PIMs, vWF might play a role in pulmonary xenograft dysfunction.

Utilizing a left single-lung transplant model, baboons depleted of anti-αGal antibodies received lungs from either vWF-deficient (n = 2); MCP-expressing (n = 5); MCP PIM-depleted (n = 5); or vWF-deficient PIM-depleted swine (n = 3).

Two out of three of the PIM-depleted, pvWF deficient grafts survived longer than any previously reported pulmonary xenografts, including PIM-depleted xenografts expressing human complement regulatory proteins. Depletion of PIM's from vWF-deficient lungs, like depletion of PIM's from hMCP lungs, resulted in abrogation of the coagulopathy associated with pulmonary xenotransplantation.

Thus, in terms of pulmonary graft survival, control of adverse reactions involving pvWF appears to be equally or even more important than is complement regulation using hMCP expression. However, based on the rapid failure of PIM-sufficient, pvWF-deficient pulmonary xenografts, pVWF-deficient pulmonary xenografts appear to be particularly sensitive to macrophage-mediated damage. These data provide initial evidence that vWF plays a role in the ‘delayed’ (24 h) dysfunction observed in pulmonary xenotransplantation using PIM depleted hMCP organs.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In pulmonary xenotransplantation, hyperacute dysfunction is characterized by rapid development of pulmonary edema, loss of compliance and functional gas exchange and marked increase of pulmonary vascular resistance within minutes to hours following reperfusion (1–8). In addition, activation of the coagulation cascade results in a consumptive coagulopathy that is reversible upon explant of the xenogeneic lung. The histology of this injury is characterized by hemorrhagic pulmonary edema and microvascular thrombosis with fibrin and platelet deposition.

Disseminated intravascular coagulation (DIC) has been proposed as the ‘third’ barrier to xenotransplantation (9), with xenoreactive antibodies and complement incompatibility being the first two. DIC is observed within minutes to hours of pulmonary xenograft reperfusion (10). Several incompatibilities between the human and porcine coagulation systems have been identified as potential underlying causes of the coagulopathy associated with pulmonary xenotransplantation. One potential problem is that porcine von Willebrand Factor (vWF) interacts in an aberrant fashion with human platelets. Von Willebrand Factor is a glycoprotein stored by platelets and endothelial cells that is released upon activation of those cells (11–13). In normal individuals, vWF binds to GPIb on platelets only if the platelets are subjected to shear stress (14,15), resulting in platelet activation and adhesion (16–19). In contrast, porcine von Willebrand Factor (pvWF) binds human (or primate) GPIb on quiescent platelets, leading to platelet aggregation even in the absence of shear stress (20,21). Thus, aberrant interactions between pvWF and human or primate GPIb could lead to widespread activation of the coagulation system, resulting in DIC. The interaction between pvWF and human GPIb may be particularly important in pulmonary xenotransplantation, as evidence suggests that pulmonary xenografts shed more pvWF than either heart or kidney xenografts (22).

Swine lungs differ from primate lungs because, in addition to the resident pulmonary alveolar macrophages, there are pulmonary intravascular macrophages (PIMs) that comprise more than 16% of microvascular surface area (23). The physiologic function of these cells is to filter blood of any foreign material or bacteria passing through the lung, a process which normally takes place in the liver or spleen in species without large populations of PIMs (24–27). In addition to their scavenging function, PIMs, produce arachidonate metabolites including thromboxane, cytokines including IL-1, IL-2, and TNF- α, and procoagulant factors, including tissue factor and PAI-1 (27–29). In acute lung injury and some xenotransplant models, evidence suggest PIMs may contribute significantly to the rapid development of pulmonary hypertension and edema (24,26,30).

Using a technique to deplete macrophages using liposomal clodronate developed by Van Rooijen (31,32), Staub was able to demonstrate in PIM depleted versus nondepleted sheep, LPS infusion resulted in complete abrogation of increases in PVR and >90% attenuation of the capillary leak (33,34).

Pierson and colleagues, using lungs from swine treated with liposomal clodronate for their heterologous perfusion experiments, found decreased production of thromboxane and preservation of a PVR comparable to the homologous control perfusion. In addition, PIM depletion decreased platelet sequestration, C3a levels, TNF-α release, and prolonged pulmonary function (29). More recently, Cantu and associates, using an in vivo pig to primate orthotopic lung transplant model with PIM depletion, were able to abrogate the consumptive coagulopathy seen in controls and extend graft function to 24 h (35). Having determined the importance of pulmonary intravascular macrophages (PIMs) in pulmonary xenotransplantation, we evaluated whether, in the absence of PIMs, vWF might play a role in pulmonary xenograft dysfunction.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Animals

All experiments were approved by the Duke University Institutional Animal Care and Use Committee and care was in accordance with National Institutes of Health (NIH) and National Society of Medical Research guidelines.

Adult (18–24 kg) baboons (Papio Anubis) were provided by Southwest Research, San Antonio, TX. Adult swine (15–30 kg) transgenic for the human complement regulatory protein membrane cofactor protein (CD46) were supplied by Nextran (Princeton, NJ). Adult swine (15–30 kg) deficient in vWF (vWFd) were obtained from The Francis Owen Blood Research Laboratory (FOBRL; Chapel Hill, NC).

Study design

Single left lung orthotopic xenotransplants were performed using the following experimental groups. Group 1 consisted of xenotransplantation of swine vWFd donor lungs in immunodepleted baboons (n = 2); Group 2 consisted of CD46 transgenic donor swine in immunodepleted baboons (n = 5); Group 3 consisted of macrophage depleted CD46 transgenic donor swine in immunodepleted baboons (n = 5); and Group 4 consisted of macrophage depleted vWFd donor swine in immunodepleted baboons (n = 3). Results from all animals in Group 2 and three of five animals in Group 3 were previously reported in a manuscript published by this lab (35). The immunosuppressive regimen and transplant operations were conduced as previously described by this laboratory (35). Porcine macrophages were depleted from porcine lungs using liposomal clodronate administered intravenously in four doses over 2 days preceding the transplant, as described previously (36,38). Graft failure was defined as a decrease in graft flow below 50 mL/min. Because we maintain blood flow through the native right lung in all of our transplant recipients, we have defined graft survival based on blood flow through the xenograft rather than the ability of the xenograft to support the recipient. This allows us to obtain a sensitive measure by which we can evaluate experimental parameters in graft function, including end-stage pathologic and immunologic processes.

Immunodepletion

Immunoglobulin depletion was accomplished as described previously either by adsorption of anti-Galα1-3Gal antibodies using a column or by blocking the anti-Galα1-3Gal antibodies using a soluble polymer (Galα1-3Gal-conjugated polyethylene glycol) (37,38). vWFd/PIM+ and hMCP/PIM+ recipients were immunodepleted using column pheresis and hMCP/PIM− and vWFd/PIM− recipients received the Gal-PEG conjugate. The immunoadsorption columns (38) and the infusion of αGal-PEG (37) both eliminate or block 98–99% of anti-Gal IgM, the predominant antibody involved in hyperacute humoral responses against porcine tissue in baboons.

Measurement of coagulation parameters

Prothrombin time, aPTT, fibrinogen levels and D-Dimer levels were assessed by the Hematology Laboratory of Antech Diagnostics (Farmingdale, NY). Platelet counts were assayed using a Pentra 60 C+ analyzer (ABX Diagnostics, Montpellier, France). TAT levels were measured using the Enzygnost TAT micro ELISA kit (Dade Behring, Marburg, Germany). Levels of PAI-1 and baboon TM were measured by ELISA using kits purchased from American Diagnostica, Inc. (Greenwich, CT).

Quantification of complement activation and measurement of antibody levels

Determination of baboon anti-Galα1-3Gal IgM and IgG levels was performed using an ELISA as previously described (39). C3a des-Arg, C5b-9 and circulating immune complex levels were measured using a commercially available ELISA kit (Quidel, San Diego, CA). C4 nephelometry determination was made using a Dade Behring BNII instrument (Deerfield, IL).

Statistics

All values are expressed as means ± SE. Survival Curve comparisons were made using the logrank test. Comparisons between two means were calculated by Student's unpaired t-test and multiple means by one-way ANOVA with the Newman–Keuls Multiple Comparison Test. Statistical significance was defined as p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Xenograft survival

Hyperacute dysfunction was observed in vWFd/PIM+ xenografts between 5 and 6 h (5.5 ± 0.71 h) following reperfusion as determined by a decreased LPAQ (Figure 1). While hMCP/PIM+ and hMCP/PIM− recipients experienced dysfunction between 0 and 9.5 h (5.6 ± 2.04 h) and 20 and 24 h (22.6 ± 1.49 h), respectively. Graft dysfunction was defined as LPAQ below 50 mL/min. On the other hand, vWFd/PIM− xenografts maintained flow significantly longer, with a mean survival time of 68.08 ± 18.28 h (range 19–109 h). Survival curve analysis demonstrated significant differences between curves (Figure 1, p = 0.004) and direct comparison between hMCP/PIM− xenografts and vWFd/PIM− xenografts demonstrated that, of all the PIM depleted xenografts, those two out of three lacking vWF survived longer than did any of those that were hMCP transgenic.

image

Figure 1. Duration of graft survival as a function of transgenic expression of a human complement regulatory protein (hMCP) or deficiency of vWF (vWFd), both with and without depletion of PIMs. Graft survival was determined to be the duration in which a flow of >50 mL/min was maintained. vWFd/PIM+; immunodepleted baboons received vWFd porcine lungs. hMCP/PIM+;, immunodepleted baboons received CD46 transgenic porcine lungs. hMCP/PIM–; immunodepleted baboons received macrophage depleted CD46 transgenic porcine lungs. vWFd/PIM− and immunodepleted baboons received macrophage depleted vWFd porcine lungs (p = 0.004).

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Pulmonary vascular resistance

Following reperfusion, xenografts in baboons receiving PIM-depleted lungs demonstrated less increase in PVR on average than did xenografts in baboons receiving lungs containing normal amounts of PIMs (Figure 2). Interestingly, the PIM-sufficient vWFd group also had a lower pulmonary vascular resistance. Whether this is a consequence of the hemodynamic perturbation seen in this group or vWFd itself is not clear. Although considerable variation in PVR was noted within groups and especially in baboons receiving PIM sufficient lungs, the PVR was significantly (p = 0.0001) different between groups. Over the course of the experiments, PIM-depleted vWFd xenografts maintained significantly lower PVR than all other groups. Specifically, PIM depleted vWFd xenografts on average maintained a resistance of 547 ± 186 dynes/cm/sec–5, whereas hMCP/PIM–, hMCP/PIM+ and vWFd/PIM+ xenografts maintained resistances of 1836 ± 309, 4741 ± 1394 and 1268 ± 1747 dynes/cm/sec–5, respectively.

image

Figure 2. Pulmonary vascular resistance (PVR) as a function of transgenic expression of a human complement regulatory protein (hMCP) or deficiency of vWF (vWFd), both with and without depletion of PIMs (p = 0.0001). The PVR was assessed as previously described by this laboratory (52).

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Hemodynamics

As previously reported (35), baboons receiving PIM-sufficient lungs experienced profound hypotension and required inotropic support (epinephrine 1 ug/min) to maintain cardiac function. The average mean arterial pressure (MAP) in these baboons dropped to as low as 30 mmHg within 60 min of reperfusion. Consistent with prior studies, such hemodynamic instability was not observed in baboons receiving PIM depleted lungs: MAPs of 78 ± 2.8 and 85 ± 5.7 mmHg following 60 min of reperfusion were observed in animals receiving hMCP/PIM− and vWFd/PIM− xenografts, respectively (Figure 3A). Thus, consistent with previous results, the presence of PIMs affected the MAP within 60 min of reperfusion (p = 0.001). These differences existed despite the inotropic support of baboons receiving PIM sufficient lungs. Even among those recipients with PIM depletion, there existed significant differences between MAPs throughout the experiment: hMCP/PIM− recipients maintained pressures of 76.18 ± 8.2 while vWFd/PIM− recipients had pressures of 87.29 ± 9.8 (p = 0.001).

imageimage

Figure 3. Hemodynamic stability of the recipients as a function of transgenic expression of a human complement regulatory protein (hMCP) or deficiency of vWF (vWFd) in the donor organ, both with and without depletion of donor PIMs. (A) Mean arterial pressure (MAP) (p = 0.001) and (B) heart rate (p = 0.0001).

An analysis of the heart rate of xenograft recipients revealed that animals receiving vWFd/PIM− organs experienced less tachycardia than did animals in other groups (p = 0.0001). Mean heart rate for vWFd/PIM− animals was 106.5 ±0.6 versus 122.6 ± 6.5, 109.0 ± 1.3 and 117.4 ± 3.7 for vWFd/PIM+, hMCP/PIM+ and hMCP/PIM− groups, respectively (Figure 3B). Direct comparison between hMCP/PIM− and vWFd/PIM–, however, demonstrated no statistically significant difference. These findings might suggest that porcine vWF contributes to the hemodynamic instability associated with pulmonary xenotransplantation, although additional work to address this issue may be warranted.

Histology

After 4 h of perfusion, patchy thrombus formation and increased cellularity were evident in all xenografts except vWFd/PIM+ grafts (Figure 4A–D). Consistent with published results, vWFd/PIM+ grafts experienced profound parenchymal and alveolar hemorrhage. Further, all xenografts demonstrated increasing neutrophil margination, migration, and sequestration. With the exception of vWFd/PIM+ grafts, all xenografts ultimately demonstrated similar pathology, which included diffuse microvascular thrombosis, increased cellularity in the form of neutrophils and pulmonary edema (Figures 4B, E and G).

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Figure 4. Histology of porcine pulmonary xenografts expressing a human complement regulatory protein (hMCP) or deficient in vWF (vWFd), both with and without depletion of PIMs. Sections were cut 4-μm thick and stained with hematoxylin and eosin. All images are shown at 20× magnification.

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Coagulation parameters

Steady decreases in platelet counts were observed in all baboons evaluated (Figure 5A). Baboons receiving PIM sufficient lungs experienced a precipitous drop in counts 15 min after reperfusion and then experienced a steady decline similar to that seen in baboons receiving PIM deficient lungs. Although the trend was for decreased platelet counts in PIM sufficient baboons, it did not reach statistical significance (p = 0.11). Platelet counts at 0, 30 min, 6 and 24 h were 278, 275, 206 and 220 thousand/mm3 for the vWFd/PIM− animals. Taken together, this suggests that factors other than vWF are involved in platelet consumption.

imageimageimage

Figure 5. Coagulation parameters of the recipients as a function of transgenic expression of a human complement regulatory protein (hMCP) or deficiency of vWF (vWFd) in the donor organ, both with and without depletion of donor PIMs. A) Platelet count in the blood of recipients (p = 0.11) B) Fibrinogen concentration in the plasma of recipients (vWFd/PIM +, p < 0.01; hMCP/PIM+, p < 0.01; hMCP/PIM–, p > 0.05) C) Thrombin-anti-thrombin complex (TAT) in the plasma of recipients (p < 0.0001).

PIM depletion with vWF deficiency together prevented consumption of fibrinogen, while baboons receiving hMCP/PIM+ lungs experienced precipitous and continued declines in fibrinogen (Figure 5B). This decrease occurred immediately after reperfusion. Interestingly, the vWFd/PIM− recipients had less perturbed levels as compared to vWFd/PIM+ (p < 0.01) and hMCP/PIM+ recipients (p < 0.01) but not hMCP/PIM− recipients (p > 0.05).

Perhaps surprisingly, PIM depleted, vWFd graft recipients exhibited significant increases in TAT generation after graft reperfusion, whereas PIM sufficient graft recipients demonstrated substantially less increases (Figure 5C; p < 0.0001); however, vWF partially mitigated this response in PIM− grafts, since TAT generation was less in animals receiving hMCP/PIM− lungs than in those receiving vWFd/PIM− lungs (p > 0.001). There was no statistically significant difference in TAT generation between groups receiving PIM sufficient lungs, regardless of vWFd or hMCP expression (p > 0.05).

Endothelial cell activation

In order to better characterize the contribution of endothelial cells (ECs) to graft failure, we examined two coagulation modulators (TM and PAI-1) that can be released by ECs. Thrombomodulin is an endothelial cell derived coagulation modulator that enhances the ability of activated protein C to prevent thrombin and clot formation (40).

All recipients experienced increases in serum recipient (baboon) TM levels except the vWFd/PIM+ group, which maintained relatively stable levels of serum TM (Figure 6A). Increases in baseline values ranged from 1.11- to 4.5-fold in the vWFd/PIM+ group (starting at 0.25 ± 0.11 and rising to 0.28 ± 0.0 ng/mL) and hMCP/PIM+ group (starting at 1.31 ± 0.12 and rising to 5.84 ± 0.43 ng/mL), respectively. The hMCP/PIM− lung recipients and vWFd/PIM− deficient recipients experienced similar increases in serum TM levels; a 3.5- to 3.6-fold increase from baseline (starting at 0.94 ± 1.09 and rising to 3.30 ± 4.73 ng/mL and starting at 1.35 ± 1.38 and rising to 4.87 ± 0.78 ng/mL, respectively). Differences in serum TM levels between groups were significant only between the vWFd/PIM+ group and all other groups (p = 0.024). This finding suggests some degree of endothelial activation outside of the donor graft, since the TM assessed in this study was likely of recipient origin. Consistent with this observation, we have not infrequently observed some gross pathology (clot formation) associated with the native recipient lung (right lung) in many pulmonary xenograft recipients.

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Figure 6. Markers of endothelial cell activation in the serum of recipients as a function of transgenic expression of a human complement regulatory protein (hMCP) or deficiency of vWF (vWFd) in the donor organ, both with and without depletion of donor PIMs. (A) Recipient (baboon) Thrombomodulin(TM) (p = 0.024) and (B) Plasminogen activator inhibitor (PAI-1) (hMCP/PIM+, p = 0.01; hMCP/PIM–, p = 0.01; vWFd/PIM–, p = 0.001).

PAI-1 is an endothelial cell protein that inhibits the formation of plasmin, preventing fibrinolysis (41). A much greater increase in the levels of PAI-1 was observed in the plasma of baboons receiving vWFd/PIM+ lungs in comparison to the plasma of hMCP/PIM+, hMCP/PIM–, and vWFd/PIM− lung recipients (Figure 6B; p = 0.01, 0.01, 0.001, respectively). These results may reflect a compensatory endothelial response in the face of profound parenchymal and airway hemorrhage seen in the vWFd/PIM+ group. No further statistically significant differences in levels of PAI-1 were noted among the other groups.

Complement activation

In order to determine what contribution complement activation might play to graft loss, serum levels of C3a des-Arg, a relatively stable metabolite of C3, was measured. Pretransplant, baboons receiving hMCP/PIM+ lungs had increased complement levels as compared to baboons receiving hMCP/PIM− and vWFd/PIM/- lungs (654.31 ± 664.33 ng/mL versus 247.14 ± 160.45 and 413.35 ± 60.54 ng/mL respectively; Figure 7). This is likely a consequence of column immunoabsorption, which has previously been shown to activate complement (6,42). Immediately following reperfusion there was an early peak in complement activation in the hMCP/PIM− and vWFd/PIM− lung recipients of 1133.18 ± 502.69 and 679.92 ± 432.67 ng/mL, respectively. It is likely that a similar peak was present in the hMCP/PIM+ group as well, although samples were not available for evaluation. Despite the absence of an early increase of C3a des-Arg in the hMCP/PIM+ group, there was significantly elevated C3a des Arg levels in the hMCP/PIM+ group compared to the levels in the hMCP/PIM− and vWFd/PIM− groups (p = 0.0257) at points beyond 1 h. Interestingly, there were no differences between the PIM depleted groups; i.e. activation of complement was similar in PIM depleted lungs, regardless of the presence of hMCP or of vWF deficiency. Sufficient serum was not available from the vWFd/PIM+ group to perform an analysis of C3a des-Arg in that group.

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Figure 7. Complement activation as a function of transgenic expression of a human complement regulatory protein (hMCP) with and without depletion of PIMs and of vWF deficiency (vWFd) with PIM depletion (p = 0.026). Levels of C3a des Arg were used as a surrogate for activation of complement Levels of C3a des Arg were not assessed in recipients of vWFd/PIM+ lungs. (inset) Levels of C4 in the serum of recipients of PIM− lungs were utilized as a measure of complement consumption in those animals: The black bars indicate C4 levels in the vWFd/PIM− group and the black bars represent C4 levels in the hMCP/PIM− group. Levels of C4 are shown both before reperfusion (Time = 0) and at 4 h post-reperfusion (p = 0.054).

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In order to exclude the possibility that consumption of complement occurred, the level of C4 in the plasma was obtained in the PIM depleted groups. No significant differences in complement levels between the pre- and post-reperfusion samples were observed in either group (p = 0.5368; Figure 7, inset).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Coagulant activation and consumption of clotting factors is a profound barrier to pulmonary xenotransplantation. In this study, we have demonstrated that coagulant and inflammatory modulation through PIM depletion and pvWF deficiency can yield in significantly longer survival than coagulant (pvWF) modulation, inflammatory modulation (PIM depletion), or complement modulation (hMCP transgene expression) alone. Perhaps unexpectedly, simultaneous modulation of coagulation and inflammation (vWFd/PIM− group) was as least as effective a simultaneous modulation of complement activation and inflammation (hMCP/PIM− group). Together, coagulant and inflammatory modulation (vWFd/PIM− group) result in less pulmonary vascular constriction and systemic hypotension, which apparently leads to prolonged pulmonary xenograft survival. These data provide strong support for the idea that pvWF plays a key role in the ‘delayed’ pulmonary xenograft dysfunction that occurs at about 24 h post-reperfusion in PIM depleted, hMCP-expressing organs).

Interestingly, vWF deficiency alone without PIM depletion was not protective and resulted in graft loss at about 5 h. This rapid loss is similar to that seen with PIM–sufficient hMCP transgenic lungs. Though both strategies yield graft failure at about 5 h, they suggest that both the inflammatory and coagulant components of hyperacute pulmonary graft dysfunction are readily capable of causing graft loss. Therefore, any strategy to prolong pulmonary xenograft function must address both components.

With identical PIM depletion and antibody control, comparison of hMCP transgenic to vWFd donor lungs reveals similar patterns of injury after transplant, with diffuse microangiopathic thrombosis, increased cellularity (primarily PMNs) and pulmonary edema. Though survival is significantly prolonged in vWF deficient PIM depleted graft recipients when compared to hMCP transgenic PIM deficient graft recipients, the mechanism of failure is not entirely clear, prompting our investigation of complement activation in this system. Though no complement regulation was performed on the vWFd/PIM− recipients other than antibody modulation, complement activation was noted to be similar between the PIM deficient groups; human MCP transgene expressing grafts fared no better than vWF deficient grafts with respect to complement activation as measured by C3a des-Arg concentration. One possible explanation for this observation is that pulmonary xenografts shed vWF in large quantities after graft reperfusion (22). Xenoreactive antibodies, in turn, may bind shed vWF, thus activating complement in the fluid phase. MCP may be an inefficient regulator of such complement activation because it is membrane bound, with limited access in the vascular space. Although speculative, it is possible that any increase in complement activation resulting from the absence of a complement regulatory protein in the vWFd lungs is offset by a decrease in complement activation resulting from decreased immune complex formation as a result of vWF deficiency. Additionally, activation of platelets has been found to lead directly to activation of the complement system on the surface of activated platelets (44). Thus, decreased levels of platelet activation due to an absence of vWF may lead directly to decreased complement activation in transplants using vWFd lungs.

As shown in previous reports, there is an absence of a consumptive coagulopathy in the PIM deficient groups (35). Comparable antibody concentration and complement activation between groups in this study suggests coagulopathy is not directly consequent to antibody and complement. In culture, macrophage cell lines have been shown to express TF after activation by LPS, TNFa, IL-1 and oxidative stress (45–50). Comparison of baboon IL-6 concentration in vWFd/PIM− to hMCP/PIM− recipients shows 2.5 times the increase from baseline in 4 h after transplant (data not shown). IL-6 is noted to increase expression of procoagulant proteins in endothelial cells (51). Increased levels of IL-6 are also associated with increased expression of soluble TF in blood (52). Bogdanov et al. have suggested that soluble TF may contribute to the thrombogenicity of extracellular fluids (53). Szotowski and co-workers confirmed that endothelial derived soluble TF (sTF) released in response to inflammatory cytokines is procoagulant in the presence of phospholipids (54). In our model, comparison between sTF in vWFd/PIM− versus hMCP/PIM− lung recipients demonstrated significantly higher levels at 3, 6, 12, and 24 h in the vWFd/PIM− lung recipients (data not shown). These findings are consistent with the idea that the coagulopathy present in the pulmonary xenografts is not a direct result of antibody and complement-mediated effects.

Despite depletion of PIMs, both hMCP and vWFd xenografts ultimately failed from thrombotic complications. The possible reasons for thrombosis are numerous. It is possible the interplay of platelets, endothelial cells and incompatibilities of anti-coagulant modulators are responsible (10). However, it must be noted that aberrant interactions between pvWF and the recipient's platelets are a major factor contributing to the early decreased graft flow seen in pulmonary xenotransplantation.

Unlike other organ xenografts, pulmonary xenografts are unique in that early failure is catastrophic secondary to diffuse coagulation and vasoconstriction. With the abrogation of hyperacute dysfunction, ‘delayed’ graft loss (24 h to 5 days) seems more closely related to endothelial activation as demonstrated by increased TAT generation and PAI-1 release. In other words, when the aberrant platelet/vWF interactions are controlled and the inflammatory component reduced by depletion of the PIMs, it seems likely that the endothelium is responsible for driving the procoagulant environment. In vitro studies have demonstrated that such activation results in increased TF expression and loss of heparan sulfate, thrombomodulin, and ATP diphosphohydrolases from the endothelial cell surface and thus a more coagulant phenotype (55–59). These findings together with species restricted regulator proteins (TF, TM) (60,61) may explain the late thrombosis seen in our model.

Some limitations are inherent in this study. First, xenoreactive antibodies were blocked in baboons receiving PIM-depleted lungs using a soluble Gal-PEG conjugate, whereas xenoreactive antibodies in baboons receiving PIM-sufficient lungs were removed in an immunoabsorptive column. Both of these techniques result in the depletion of 98–99% of xenoreactive IgM, the predominant antibody in baboon serum responsible for hyperacute rejection. Thus, while it is unlikely that the dysfunction of the PIM-sufficient grafts was due to high levels of antibody, it is possible that the use of the column resulted in the production of anaphylatoxins, or the like, which led to the dysfunction of these grafts. This seems unlikely given that the column depletion technique has no effect on the native lung and has been used successfully by others without evidence of lung-related injuries. A second limitation is that a number of residual lung macrophages might lie within the bronchus-associated lymphoid tissue (BALT) or perhaps are compartmentalized on the alveolar side of the lung (pulmonary alveolar macrophages; PAMs). The fact that the pathology of graft failure is similar but delayed in PIM− organs when compared to PIM+ organs argues in favor of the idea that residual macrophages play a key role in the graft failure of even PIM depleted organs. A third limitation is that our definition of xenograft survival does not predict the life-sustaining capacity of this xenograft, nor does it reflect the pathobiology seen with physiologic transpulmonary blood flow. Improved ways of defining xenograft function under biologically relevant conditions include flow through the xenograft approximating 50% of cardiac output, or, ideally, life-supporting function of the xenograft with native lung occlusion.

Work with Gal knockout pigs suggests that deletion of Gal may eliminate the aberrant interaction between pvWF and primate platelet GPIb (62). This may obviate the need for vWFd swine in xenotransplantion and highlights the fact that Gal elimination might be important in graft survival, regardless of the presence of xenoreactive antibody. Further studies in this area are eagerly anticipated. Further, greater characterization of the molecular events of late thrombosis seen in this model is also needed. Although we are far from human clinical trials, this study has demonstrated significant improvement in pulmonary xenograft survival and suggests new targets for future study.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors thank George Quick, Ronnie Johnson, MaryLou Everett, Michael Lowe and Elaine Parker for their assistance with this project. This work was supported by NIH grant R01 HL60232-03 and F32 HL 71457-01.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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